Voltage-gated sodium channels (VGSC or Nav) produce the rapid upstroke of the action potential and are important elements for maintaining electrical excitability and assuring the coordination of excitation-contraction coupling in striated muscle and neuronal excitability. As shown in FIG. 1, they are composed of one α-subunit (260 kDa in the example shown), which forms the core of the channel and which is responsible for the voltage-dependent gating and ion permeation (Catterall W A, Annu Rev Biochem 1986, 55: 953-985; Fozzard H A and Hanck D A, Physiol Rev 1996, 76: 887-926; Armstrong C M and Hille B, Neuron 1998, 20: 371-380). The α-subunit is composed of four homologous domains (DI-DIV), each with six α-helical transmembrane-spanning segments (S1-S6). The S1-S4 domains form the voltage sensor domains (Stuhmer W et al., Nature 1989, 339: 597-603; Yang N et al., Biophys J 1997, 73: 2260-2268; Kontis K J et al., J Gen Physiol 1997, 110: 391-401). The short linkers connecting the S5 and S6 segments form the external mouth of the pore and the selective filter (Pérez-Garcia M T et al., Biophys J 1997, 72: 989-996; Yamagishi T et al., Biophys J 1997, 73: 195-204; Chiamvimonvat N et al., Neuron 1996, 16: 1037-1047; Pérez-Garcia M T et al., Proc Natl Acad Sci USA 1996, 93: 300-304). The cytoplasmic linker between the third (DIII) and fourth (DIV) homologous domains acts as a “hinged lid” that occludes the internal end of the permeation pathway during inactivation (Stühmer W et al., 1989, supra; Armstrong C M and Bezanilla F, J Gen Physiol 1977, 70: 567-590; West J W et al., Proc Natl Acad Sci USA 1992, 89: 10910-10914). Residues of the S6 segments from each of the four homologous domains (DIS6-DIVS6) line the internal vestibule and contribute to the binding site for local anaesthetics (LA) and antiarrhythmic drugs (Ragsdale D S et al., Proc Natl Acad Sci USA 1996, 93: 9270-9275). The cytoplasmic ends of the S6 segments and the short linkers from each of the four homologous domains that connect the S4-S5 segments contribute to the binding site for the native inactivation gate (Smith M R and Goldin A L, Biophys J 1997, 73: 1885-1895; McPhee J C et al., Proc Natl Acad Sci USA 1994, 91: 12346-12350; McPhee J C et al., J Biol Chem 1995, 270: 12025-12034).
Structure-function studies indicated that the S5-S6 linkers constitute the pore-forming regions known as P-loops of the channel (Pérez-Garcia M T et al., 1996, supra; Heinemann S H et al., Nature 1992, 356: 441-443; Terlau H et al., FEBS Lett 1991, 293: 93-96). Each P loop is composed of two short segments called SS1 and SS2, for short segment 1 and short segment 2 respectively, they span part of the plasma membrane (Terlau H et al., 1991, supra; Guy H R and Conti F, Trends Neurosci 1990, 13: 201-206; Guy H R and Seetharamulu P, Proc Natl Aced Sci USA 1986, 83: 508-512).
Sodium (Na) channel blockers have been developed and used for therapeutic purposes for several decades. One of the earliest compounds used for therapeutic purposes, that was later shown to block Na channels is cocaine. Cocaine, an aminoester, was the first local anesthetic drug useful in clinical surgery but it had undesirable side effects. It was however soon realized that the anesthetic properties of cocaine were preserved in chemically similar structures that had less undesirable side effects. This quickly led to the development of an entire class of cocaine-related compounds comprising other aminoesters like benzocaine and procaine, as well as aminoamides, like bupivacaine and lidocaine. Most of these drugs were/are typically not administered orally, but topically or intrathecally, thereby preventing adverse side effects, like convulsions and cardiovascular collapse, still associated with these drugs when applied systemically. The mechanisms by which these compounds interact with Na channels have been the subject of many scientific studies. Lidocaine in particular, still widely used in the clinic today, has been studied extensively. Lidocaine has shown efficacy in numerous pain conditions including diabetic neuropathy and postherpetic neuralgia.
Besides the “caine” class of analgesic compounds, certain clinically used anticonvulsants, antidepressants, and antiarrythmics have inhibitory activity on Na channels, which at least partially, underlie their clinical efficacy. Among such compounds are phenytoin, carbamazepine, and aminotryptyline. All these drugs block Na channels by binding to the DI-IV S6 transmembrane helices.
More recently, a number of mutations have been found to cause abnormal Na channel functions leading to human diseases or Na channelopathies such as periodic paralysis, myotonia, long QT syndrome and other cardiac conductance disturbances, pain, and epilepsy George A L Jr., J Clin Invest 2005, 115: 1990-1999).
Conventional methods for assaying sodium channel activity include radiolabeled toxin-binding assays, radioactive ion influx assays, electrophysiological patch-clamp assays, and membrane potential dyes (Reviewed by Terstappen, 2005, Drug Discov Today: Technologies 2(2): 133-40). All these assays have major disadvantages that limit their use. For example: i) the radioactive ion influx method requires long incubation time and multiple wash steps, necessitating non-homogeneous assay format. Moreover, it requires the use of chemical modifier of channel inactivation, introducing the risk of false positive or false negative results, and finally, it produces a large quantity of costly radioactive waste; ii) The patch clamp technique, largely considered the “gold standard”, has inherent limitations, including low throughput and specialized equipment incompatible with standard laboratory robotics. Although higher throughput can be achieved with higher throughput patch-clamp such as IonWorks™ or PatchXpress™ (both from Molecular Devices Inc.), these assays are still relatively expensive and not well adapted for fast kinetics of VGSC.
Currently, in the industry, another approach that is used for drug-screening assays with VGSC is based on membrane potential-sensitive fluorescent dyes, such as bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), because there are no efficient sodium dyes available. However, three major problems are associated with this technology: (i) Dyes such as DiBAC4(3) are sensible to any membrane potential changes and as a result it is not possible to employ extracellular potassium to open sodium channels and measure the sodium influx. It is necessary to use toxins, such as veratridine which the mechanism of action is not yet elucidated, to activate the sodium channel and monitor the ion influx. In a high-throughput screening context, this can generate many false positive or false negative results because the direct binding of veratridine to channels clearly changes their native conformation, and probably alters the interaction with the compounds to be tested; (ii) the use of fluorescent dyes involves time-consuming wash steps as well as the loss of cells and signal; (iii) membrane potential-sensitive fluorescent dyes are expensive, and are not suitable for endogenous expression due to their low response time and low sensitivity (Reviewed by Terstappen, 2005, supra).
There is thus a need for the development of novel reagents and methods for the identification of sodium channel modulators.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.